Pajovic-Šcepanovic et al.

Flavan-3-ols of Montenegrin grape cultivars 1

Suitability of flavan-3-ol analysis to differentiate grapes from Vranac,

Kratošija and Cabernet Sauvignon (Vitis vinifera L.) grown in Montenegro
 CEPANOVI
R. PAJOVIC-Š   1, S. WENDELIN2, A. FORNECK3 and R. EDER2
C
1
Department for Viticulture and Enology, Biotechnical Faculty, University of Montenegro, ME-81000, Podgorica,
Montenegro; 2 Department for viticulture and oenology, Federal College and Research Institute for Viticulture and
Pomology, A-3400, Klosterneuburg, Austria;
3
Division of Viticulture and Pomology, University of Natural Resources and Life Science, 3430, Tulln/Donau, Austria
Corresponding author: Dr Reinhard Eder, email reinhard.eder@weinobst.at

Abstract
Background and Aims: Interest in the production of wines from indigenous cultivars such as Vranac and Kratošija is
steadily increasing, since they offer distinct characteristics. In contrast to popular cultivars, little is known about their chemi-
cal composition, especially in Montenegro. Many important characteristics of red wines are related to phenolic substances.
Therefore, it is imperative to determine the phenolic composition of these cultivars.
Methods and Results: Flavan-3-ol monomers of seeds were analysed by reversed phase HPLC after thioacidolysis. In the
perchloric extract of skins, flavan-3-ol monomers and dimers were determined by HPLC. Catechin represented the most
abundant flavan-3-ol in seeds, with a high concentration of epicatechin-3-O-gallate also found in Vranac. In the skins,
procyanidin B1 and catechin were the most abundant phenolic substances. Using multivariate statistics, a unique profile of
the ratio of flavan-3-ols from each cultivar was found.
Conclusions: Cultivar and geographical origin considerably influenced the flavan-3-ol composition of the three cultivars.
The flavan-3-ol concentration of berries exhibited characteristic varietal differences, with the order of concentration being
Cabernet Sauvignon > Kratošija > Vranac. The indigenous Montenegrin cultivars had a significantly lower proportion of
seed and skin mass than that of Cabernet Sauvignon, which resulted in a lower flavan-3-ol concentration in the berries.
Significance of the Study: The flavan-3-ol concentration of grapes from Vranac, Kratošija and Cabernet Sauvignon culti-
vars grown in Montenegro was determined for the first time. These results further broaden knowledge relating to characteri-
sation of wine, optimisation of its production and authenticity control.

Keywords: catechin, flavan-3-ol composition, grape, phenolic substance, proanthocyanidins

Introduction seeds at a concentration higher than in the skins (Bourzeix

Phenolic substances play an important role in the quality of
grapes and wine. They include the flavan-3-ols, which are
considered essential constituents of red wine. Flavan-3-ols
are primarily monomers of (+)-catechin and (−)-epicatechin,
and to a lesser extent, epigallocatechin, gallocatechin and
epicatechin-3-O-gallate. Flavan-3-ols also can exist as dimers
and larger molecules. Oligomeric and polymeric forms of
flavan-3-ols are called proanthocyanidins or condensed tan-
nins (Bourzeix et al. 1986). Proanthocyanidins and con-
densed tannins are, respectively, composed of flavan-3-ol
monomers linked together [units of (+)-catechin and (−)-epi-
catechin], and of interflavonoid bonds between monomers of
gallocatechin and epigallocatechin. Overall, flavan-3-ols have
an impact on the sensory quality (astringent and bitter attri-
butes) of wine, and are important for ageing and colour sta-
bility (Somers 1971). Monomers are more bitter than
astringent, whereas proanthocyanidins are more astringent
and less bitter (Peleg et al. 1999, Chira et al. 2009).
The flavan-3-ols of wine have attracted considerable inter-
est because of their antioxidant properties and importance for
human health (Rigo et al. 2000). In grape berries, flavan-3-ols
are present mainly in skins and seeds, from which they are
extracted into the must during vinification and remain in the
resulting wine (Souquet et al. 1996). They are present in grape
et al. 1986, Sun et al. 1999, De Freitas et al. 2000, Monagas
et al. 2003). The distribution of flavan-3-ols in the grape berry
varies between seed and skin (Chira et al. 2009, Mattivi et al.
2009, Pajovic et al. 2014a). Grape skin is mainly composed of
four monomers (catechin, epicatechin, gallocatechin and
epigallocatechin), procyanidins and prodelphinidin oligomers,
whereas grape seeds contain mainly three monomers (cate-
chin, epicatechin and epicatechin-3-O-gallate) and pro-
anthocyanidin oligomers (Mattivi et al. 2009).
It is generally considered that the flavan-3-ols of skins
are more easily transferred into wine (Sun et al. 1999) and
that they therefore contribute more to the flavan-3-ol com-
position of wine (Gris et al. 2011).
The biosynthesis of flavan-3-ols and proanthocyanidins
in grapes is highly specific at the cultivar level; moreover,
the absolute concentration of free flavan-3-ols in grape skins
and seeds can be used to classify cultivars (Mattivi et al.
2009). Besides the genetic factor, the accumulation of
flavan-3-ols is influenced by several other factors, such as
climate, soil, vintage, viticultural practices and yield
(Downey et al. 2006). Grapegrowing and winemaking
started in ancient times in Montenegro, and today, 96% of
the vineyards are concentrated in the Podgoricki sub-region.
According to the Geoviticulture Multicriteria Climatic

doi: 10.1111/ajgw.12406
© 2019 Australian Society of Viticulture and Oenology Inc.
2 Flavan-3-ols of Montenegrin grape cultivars
Classification System (Tonietto and Carbonneau 2004), the
weather in the Podgorica district is classified as ‘very warm’
in terms of the Huglin’s heliothermal index (HI + 3) and as
a ‘warm region’ in terms of the Winkler Index—CIII zone. It
is believed that the Podgorica vineyard district is favourable
for the cultivation of vines and consequently the production
of high-quality wines (Vujadinovic et al. 2016).
Winemaking in Montenegro is traditionally based on the
two local autochthonous red grape cultivars Vranac and
Kratošija, which are also widespread in the neighbouring
countries of Macedonia, Bosnia, Herzegovina, Croatia and
Serbia (Ulicevic 1966). These local cultivars have considerable
importance for the wine regions of the Western Balkan coun-
tries (Božinovic 2005). Kratošija dominated in the Montene-
grin vineyards until the emergence of phylloxera (Ulicevic
1966). Vranac is currently the dominant red grape cultivar,
with the area under cultivation of Kratošija decreasing, while
Cabernet Sauvignon plantings are increasing in Montenegrin,
and other wine regions in the Western Balkan countries. The
majority of previous research into autochthonous Montene-
grin cultivars concerns Vranac, which economically is the
most significant and the most abundant red cultivar in the
Western Balkan region. To date, studies into the phenolic
concentration and the distribution of low and high molecular
mass proanthocyanidins in seeds and skins of red grape culti-
vars from Montenegro have used only spectrophotometric
methods (Pajovic et al. 2014a).
Based on anthocyanin and hydroxycinnamic acid compo-
sition, a profile of the phenolic substances for Montenegrin
autochthonous cultivars and Cabernet Sauvignon was devel-
oped (Pajovic et al. 2014b). Montenegrin red wines have
recently been characterised by accurate and reliable chro-
matographic techniques (Pajovic-Šcepanovic et al. 2018).
This is the first study with chromatographic techniques of
the flavan-3-ol composition of skins and seeds of grapes from
the three most important grape cultivars in the Western Bal-
kan region. The aim of this work was to determine the flavan-
3-ol composition of grapes of the Montenegrin red cultivars
Vranac and Kratošija (Vitis vinifera L.), and of Cabernet
Sauvignon, with all grapes originating from the same vintage.
Materials and methods
Chemicals and reagents
The following chemicals were used during analysis: methanol
(HPLC grade), hydrochloric acid (37%), perchloric acid and
formic acid (Merck, Darmstadt, Germany); distilled water and
MilliQ water (Millipore, Billerica, MA, USA); and toluene-
α-thiol (99% purity) (Sigma-Aldrich, St Louis, MO, USA).
Analytical standards were sourced commercially: (+)-catechin
and (−)-epicatechin (Sigma-Aldrich); (−)-epigallocatechin,
(−)-epicatechin-3-O-gallate, procyanidin B1 and procyanidin
B2 (Extrasynthese, Genay, France); and (−)-epigallocatechin-
3-O-gallate (Roth, Karlsruhe, Germany).
Grape samples
Vitis vinifera L. cvs Vranac, Kratošija and Cabernet
Sauvignon were harvested during the 2011 vintage at tech-
nological maturity (i.e. 23–24
Brix), from six vineyards
located in the Podgorica district. The vineyard locations
were: Šipcanik (L1 42
200 50.0700 N, 19
150 46.7100 E, 27 masl);
Rogami (L2 42
280 14.2600 N, 19
150 33.2900 E, 51 masl); Kokoti
(L3 42
240 03.7300 N, 19
120 35.1100 E, 23 masl); Lješkoplje
(L4 42
240 01.4200 N, 19
120 34.9400 E, 31 masl); Nikolj Crkva
(L5 42
240 04.0500 N, 19
150 56.3500 E, 50 masl); and Kuci (L6
Australian Journal of Grape and Wine Research 2019
42
300 00.7700 N, 19
230 36.2700 E, 454 masl). The locations
Šipcanik and Nikolj Crkva (L1 and L5) are owned by the

largest viticulture company in Montenegro, Cemovsko Polje,
and comprise 60% of the total vineyard area in the Podgo-
rica district. The smallest vineyard (L6) was located at the
highest altitude. Representative grape samples (20 kg per
cultivar per location) were harvested and pooled. The physi-
cal composition of 100-berry sub-samples (selected ran-
domly and in triplicate for each cultivar, at each location)
were determined immediately after harvest. The mass of
100 berries was measured, before skins and seeds were sep-
arated from pulp, and subsequently dried on filter paper at
room temperature. The mass of skins and seeds was then
calculated as a proportion of berry mass, in order to calcu-
late flavan-3-ols as mg/kg in the skins and seeds of grapes
from each cultivar and location. One thousand berries were
taken from sampled grapes and stored at −80
C until fur-
ther analysis. Three months later, in January, samples were
transferred to −20
C for 2 days prior to extraction.
Sample preparation for extraction of phenolic substances
Berry samples were thawed for 30 min before being pre-
pared for analysis. Fifty berries were sampled and weighed
on the basis of their determined mass (Table 2). The berries
were manually separated into skins, seeds and pulp with a
spatula. Skins were blotted on paper towels to remove any
residual pulp, before the mass of the pulp, seed and skin
was determined with an analytical balance. For each culti-
var and each location, three biological replicates, each of
50 berries, were analysed.
For extraction and analysis of flavan-3-ols, skin (5 g) was
homogenised in 6% (w/v) perchloric acid in water (10 mL)
according to the modified method of Vrhovšek et al. (1997).
Perchloric acid was added to precipitate proteins and inhibit
polyphenoloxidase activity. The skin was homogenised for
10 min at 10 000 rpm with an HD 4AP homogeniser
(Edmund Bühler, Tübingen, Germany). The extract was then
filtered through a 0.45 μm membrane filter (Multiclear 13;
CS-Chromatographie Service, Langerwehe, Germany).
Seeds were prepared for thioacidolysis by drying in a vac-
uum oven at 70
C (Fisherbrand Isotemp Model 282A; Fisher
Scientific, Hampton, NH, USA). After 7 days, seeds (2 g) were
mixed with ascorbic acid (20 mg) and potassium metabisulfite
(20 mg), and milled (Mixer mill MM 400; Retsch, Haan, Ger-
many) at 1800 rpm for 7 min, to obtain dry seed powder.
Thioacidolysis
Thioacidolysis of seeds was performed according to the
Organisation Internationale de la Vigne et du Vin (2009)
method for the differentiation of proanthocyanidin tannins,
with slight modification (Návojská et al. 2012). Dried seed
powder (10 mg) was dissolved in 10 mL of methanol and
mixed with 1 mL of thioacidolysis reagent (470 μL of
toluene-α-thiol introduced into a hydrochloric acid solution
comprising 140 μL of 12 mol HCl in 10 mL of methanol) in
an hydrolysis tube. The mixture was heated (with stirring)
at 60
C for 10 min. The tube was then cooled to ambient
temperature, after which distilled water (1 mL) was added
and the resulting mixture was analysed by HPLC.
HPLC analysis of flavan-3-ols in seeds
A Hewlett-Packard series II 1090 AminoQuant chromatog-
raphy system equipped with a diode array detector (DAD)
(Hewlett Packard, Santa Clara, CA, USA) was used to
© 2019 Australian Society of Viticulture and Oenology Inc.
Pajovic-Šcepanovic et al.
determine flavan-3-ols in seed extracts. The flavan-3-ols
were separated on a reverse phase LiChrospher 100, RP-18
(250 × 4 mm) column (Merck) filled with 5 μm particles
(Organisation Internationale de la Vigne et du Vin 2009).
Instead of phosphoric acid, however, solution A consisted of
1% formic acid in MilliQ water, while solution B consisted
of 1% formic acid in methanol. The flavan-3-ols were sepa-
rated at 40
C applying the following solvent gradient: 5% B
at 0 min, 10% B at 14 min, 30% B at 34 min, 90% B at
40 min and maintained until 50 min, 5% B at 55 min and
post-run (15 min). Flow rate was constant at 1 mL/min. The
measuring wavelength was set at 280 nm and the injection
volume was 20 μL. The flavan-3-ol monomers were identi-
fied and quantified using external standards on the basis of
their retention times. The following standards were used:
(+)-catechin, (−)-epicatechin, (−)-epigallocatechin, (−)-epi-
catechin-3-O-gallate and (−)-epigallocatechin-3-O-gallate.
Limit of detection for (+)-catechin and (−)-epicatechin was
0.8 mg/L (Liszt et al. 2015).
HPLC analysis of flavan-3-ols in skins
The concentration of flavan-3-ol was determined by HPLC
according to the modified method of Vrhovšek et al. (1997).
A 1200 Agilent rapid resolution HPLC system (Agilent Tech-
nologies, Santa Clara, CA, USA) fitted with an autoinjector
(5 μL injection volume) and a DAD set at 280 nm. The
flavan-3-ols were separated with a C18 reverse phase col-
umn (Zorbax SB-C18, 150 × 2.1 mm; Agilent) filled with
1.8 μm particles, at a temperature of 40
C. Two mobile
phases were used: formic acid in water [0.5% (v/v), pH 2.3]
(solvent A) and methanol (solvent B). Gradient elution was
applied at a flow rate of 0.25 mL/min using the following
solvent gradient: 4% B at 0 min, 5% B at 30 min, 8% B at
36 min and maintained until 40 min, 12% B at 60 min,
21% B at 70 min, 70% B at 75 min and maintained until
80 min, 4% B at 87 min and post-run (15 min). The con-
centration of flavan-3-ols was determined by an external
standard method by comparison of the retention time and
UV spectrum with that of commercial standards: (+)-cate-
chin, (−)-epicatechin, procyanidin B1 and procyanidin B2.
Limit of detection for (+)-catechin and (−)-epicatechin was
0.8 mg/L and for procyanidin B1, and procyanidin B2
0.7 mg/L (Liszt et al. 2015).
Data analysis
The experimental data were analysed using the statistical
package SPSS Statistics 20 (IBM, New York, NY, USA). Dif-
ferences between the three grape cultivars and the six loca-
tions, as well as their interactions, were determined by
ANOVA and least significant difference test for an α = 5%.
Homogeneity of variances was analysed using Levene’s test.
The relationships between cultivars grown at different loca-
tions were investigated by principal component analysis
(PCA) based on the relative proportion of seven flavan-3-ols
found in grape skins and seeds. Means for three replicates
were used to create a correlation matrix from which
standardised principal component (PC) scores were
extracted. Scatter plots of the first two PCs were created
with Statistic/Graph (StatPont Technologies, Warrenton,
VA, USA). In order to determine which of the PCs
accounted for the greatest variation, the eigenvalues of the
two PCs were compared for each trait. Data were processed
with the statistical program Statistic (StatSoft, Tulsa,
OK, USA).
© 2019 Australian Society of Viticulture and Oenology Inc.
Flavan-3-ols of Montenegrin grape cultivars 3
Results
The concentration of selected flavan-3-ols present in the seeds
and skin of grapes from the three cultivars, Vranac, Kratošija
and Cabernet Sauvignon, and from six vineyard locations in
Montenegro is shown in Table 1. In the seeds, five flavan-3-ol
monomers (catechin, epicatechin, epigallocatechin, epicatechin-
3-O-gallate and epigallocatechin-3-O-gallate) were analysed
after thioacidolysis. In the perchloric extract of skins, catechin,
epicatechin and procyanidin B1 and procyanidin B2 were
determined.
Flavan-3-ols in seeds
The concentration of flavan-3-ols was generally higher in
seeds than in skins (Table 1). Of the five flavan-3-ol
monomers measured, only three were detected, being
catechin, epicatechin and epicatechin-3-O-gallate. The
average concentration of catechin and epicatechin was
highest in Cabernet Sauvignon seeds (462.2 and
330.9 mg/kg, respectively); significantly lower in
Kratošija seeds (291.1 and 187.5 mg/kg, respectively)
and the lowest in Vranac seeds (211.1 and 132.5 mg/kg,
respectively). The highest epicatechin-3-O-gallate con-
centration, however, was observed in Vranac seeds
(167.0 mg/kg), while a lower concentration was detected
in Cabernet Sauvignon seeds (141.6 mg/kg) and in
Kratošija seeds (115.0 mg/kg).
The relative concentration (as a proportion) of flavan-3-ol
monomers found in Vranac, Kratošija and Cabernet
Sauvignon seeds (averaged across the six vineyard locations) is
shown in Figure 1. Catechin represented the main flavan-3-ol
monomer in seeds and the order of concentration among cul-
tivars was Cabernet Sauvignon ≥ Kratošija ≥ Vranac. The sec-
ond most abundant flavan-3-ol monomer present in Cabernet
Sauvignon and Kratošija seeds was epicatechin, whereas for
Vranac seeds, epicatechin-3-O-gallate was the next most abun-
dant flavan-3-ol. Montenegrin red cultivars can therefore be
considered to be rich in epicatechin-3-O-gallate.
Flavan-3-ols in skin
Grape skins contained a concentration of flavan-3-ols signifi-
cantly lower than that of seeds (Table 1). The total flavan-3-ol
concentration present in Cabernet Sauvignon skins
(10.5 mg/kg) was significantly higher than that of skins of the
Kratošija (7.4 mg/kg) and Vranac (6.4 mg/kg) cultivars. The
highest catechin concentration was found in skins of Cabernet
Sauvignon (3.2 mg/kg). A significantly lower concentration
was found in Kratošija (1.8 mg/kg) and Vranac skins
(1.6 mg/kg). Catechin was the main flavan-3-ol monomer pre-
sent in the skins of the three cultivars; approximately 75% of
the flavan-3-ol monomer concentration comprised catechin.
Epicatechin was present at lower concentration, but with a sig-
nificantly higher concentration in Cabernet Sauvignon
(1.4 mg/kg) compared to that of Vranac and Kratošija
(0.8 mg/kg each). Procyanidin B1 was the main dimer, with a
larger concentration detected in the skins of the three cultivars
than for any of the other flavan-3-ols. The procyanidin B1 con-
centration of Cabernet Sauvignon skins (5.3 mg/kg) was
highest; a significantly lower concentration was found in
Kratošija (4.0 mg/kg) and Vranac (3.2 mg/kg) skins.
Procyanidin B2 was present at lower concentration, with no
significant difference observed among the three cultivars.
Physico-mechanical characteristics
The berry mass and mass of skin and seeds as a proportion
of the berry mass for each cultivar are presented in Table 2.
 4

Table 1. Concentration of flavan-3-ols in skins and seeds of grapes of the Vranac, Kratošija and Cabernet Sauvignon cultivars grown at six locations in Montenegro†.

Concentration (mg/kg)

Seeds Skins Seeds and skins

Cultivar and Epicatechin-3-

location Catechin Epicatechin O-gallate Total Catechin Epicatechin Procyanidin B1 Procyanidin B2 Total Total

Vranac
Šipcanik 181.4  32.3c 116.7  28.8b 150.9  24.9c 449.0  83.2c 1.6  0.1b 1.0  0.5a 3.0  0.6bc 0.8  0.1a 6.4  0.3b 455.2  83.3c
Rogami 160.7  31.5d 100.0  20.6bc 134.7  30.7d 395.4  82.6d 1.6  0.1bc 0.6  0.6c 3.3  0.1b 0.9  0.3a 6.4  0.2b 401.8  82.4d
Flavan-3-ols of Montenegrin grape cultivars

Kokoti 193.3  5.5c 110.5  13.7b 153.4  2.8c 457.2  21.6c 1.7  0.1a 0.9  0.9a 3.7  0.3a 0.9  0.0a 7.2  0.4a 464.4  21.6c
Lješkoplje 274.7  46.4ab 181.7  31.1a 195.8  59.1b 652.3  144.7b 1.5  0.2c 0.8  0.8ab 2.9  0.3bc 0.9  0.2a 6.0  0.2c 658.3  144.5b
Nikolj Crkva 297.8  19.7a 196.2  23.2a 235.8  16.6a 729.7  57.6a 1.7  0.1a 0.6  0.6c 3.1  0.2b 0.6  0.1 6.1  0.3c 735.8  57.4a
Kuci 158.6  41.1d 90.0  18.7c 131.5  25.5d 380.2  82.9d 1.8  0.1a 0.8  0.1ab 3.0  0.2bc 0.7  0.1ab 6.0  0.4c 387.9  83.2d
Mean 211.1  17.6C 132.5  9.4C 167.0  16.8A 510.6  43.1C 1.6  0.02B 0.8  0.8B 3.2  0.2C 0.8  0.1A 6.4  0.0C 510.0  43.0C
Kratošija
Šipcanik 288.7  23.9d 168.7  9.7c 113.0  4.6b 570.4  36.9c 2.1  0.1a 0.9  0.1a 3.7  0.3c 1.0  0.1a 7.4  0.7b 577.7  83.3c
Rogami 323.4  28.8bc 199.7  39.2b 121.1  13.4b 644.2  77.5b 1.5  0.1b 0.6  0.1c 4.7  0.5a 0.8  0.0ab 7.4  0.3b 651.6  83.2b
Kokoti 310.0  31.0c 216.7  32.7a 110.6  7.5b 637.3  70.5b 1.4  0.2b 0.7  0.1c 4.1  0.8b 0.8  0.1a 7.0  0.9c 644.3  21.6b
Lješkoplje 273.3  15.5e 169.6  15.9c 102.5  16.9bc 545.4  45.3c 2.0  0.4a 1.0  0.3a 3.0  0.2 1.1  0.3a 7.0  0.7c 552.1  144.5c
Nikolj Crkva 337.8  22.9a 230.8  9.5a 145.7  15.2a 714.4  44.5a 2.1  0.1a 1.1  0.3a 4.5  0.4a 0.9  0.1a 8.5  0.3a 722.9  82.4a
Kuci 213.2  28.2e 139.4  20.3d 97.3  14.6c 450.0  62.5d 1.9  0.1a 0.7  0.1b 4.1  0.8b 0.7  0.1b 7.1  0.4c 457.1  57.4d
Mean 291.0  13.7B 187.5  14.2B 115.0  6.3C 539.6  32.7B 1.8  0.04B 0.8  0.1B 4.0  0.1B 0.8  0.1A 7.4  0.2B 601.0  43.0B
Cabernet Sauvignon
Šipcanik 470.9  60.7b 309.6  38.8d 142.9  21.9bc 920.7  121.4b 3.4  0.1a 1.5  01.a 5.5  0.1b 1.0  0.1a 11.5  0.3a 932.2  83.3b
Rogami 386.3  27.2d 271.1  20.0e 123.1  10.6c 780.5  57.5d 3.6  0.1a 1.7  0.1a 5.8  0.1a 0.9  0.2a 12.0  0.2a 792.5  83.2d
Kokoti 544.0  27.6a 371.0  24.9b 151.5  6.5b 1066.5  37.5a 2.7  0.1c 1.2  0.1b 4.4  0.1e 0.8  0.1ab 8.8  0.5c 1075.3  21.6a
Lješkoplje 443.0  39.7d 350.7  29.4c 136.6  17.2bc 910.6  98.8b 2.9  0.2b 1.2  0.1b 5.2  0.1c 0.7  0.1b 9.9  07b 920.5  144.5b
Nikolj Crkva 523.8  101.0a 400.0  67.8a 172.4  32.1a 1096.2  200.2a 3.1  0.03b 1.1  0.1b 4.7  0.1d 0.8  0.2ab 9.7  0.3b 1105.8  82.4a
Kuci 405.0  51.9d 305.2  47.3d 123.3  15.2c 833.2  113.6c 3.3  0.1ab 1.6  0.1a 6.0  0.1a 0.9  0.0a 11.5  0.6a 844.6  57.4c
Mean 462.2  29.1A 330.9  24.6A 141.6  10.0B 934.6  63.4A 3.2  0.1A 1.4  0.1A 5.2  0.1A 0.7  0.2A 10.5  0.2A 945.2  43.0A

†Data are average values  SD (n = 3). For each cultivar, values followed by different lowercase letters in the same column are significantly different (P < 0.05) by location. Mean values represent data averaged by location and
means followed by different uppercase letters within columns are significantly different (P < 0.05) by cultivar.
Australian Journal of Grape and Wine Research 2019

© 2019 Australian Society of Viticulture and Oenology Inc.

Pajovic-Šcepanovic et al. Flavan-3-ols of Montenegrin grape cultivars 5

Figure 1. Relative concentration of the flavan-3-ols catechin ( ),

epicatechin ( ) and epicatechin-3-O-gallate ( ) in the seeds of Vranac,
Kratošija and Cabernet Sauvignon grapes.

The mass of Vranac and Kratošija berries (225 and

244 g/100 berries) was almost twice that of Cabernet
Sauvignon berries (113 g/100 berries). The mass proportion
of seeds was highest in Cabernet Sauvignon (4.9%), com-
pared to Vranac (3.2%) and Kratošija (2.2%). Cabernet
Sauvignon berries were also characterised by a high skin
proportion (14.4%), compared to berries from Vranac and Figure 2. Scatter plot showing distribution of flavan-3-ols in skin and
seeds.
Kratošija (10.3 and 9.8%, respectively).

variability observed. Correlations between the concentration

Classification of cultivars based on the pattern of
of flavan-3-ols and the first two PCs are shown in Figure 2.
flavon-3-ols Figure 3 shows the geometrical distances between the culti-
The individual flavan-3-ols as a proportion of the total was
vars and the six vineyard locations, as determined on the
subjected to PCA in order to determine to what extent the
basis of all analytical measures.
cultivar affected berry composition. The PCA indicated that
Based on the position of samples on the scatter plot
the first two components explained 80.46% of the total
(Figure 2) it can be observed that the grape cultivars were
separated into three distinct groups. Cabernet Sauvignon
Table 2. Berry mass and proportion of skins and seeds for grapes of the
Vranac, Kratošija and Cabernet Sauvignon cultivars grown at six locations in
Montenegro†.

Cultivar and Proportion Proportion

location Berry mass (g) skins (%)‡ seeds (%)‡

Vranac
Šipcanik 257  5.3a 8.8  0.2c 3.1  0.2bc
Rogami 190  21.8d 11.0  1.0a 2.7  0.4c
Kokoti 223  4.6b 9.8  0.3ab 3.4  0.3ba
Lješkoplje 210  3.6c 10.2  0.8ab 2.7  0.4c
Nikolj Crkva 251  5.7a 9.1  0.7bc 4.0  0.7a
Kuci 220  2.6bc 9.8  0.3bd 3.3  0.3bc
Mean 225  25.2B 9.8  0.9C 3.2  1.7B
Kratošija
Šipcanik 238  2.0c 10.6  0.7bc 2.2  0.4ab
Rogami 242  2.0bc 10.4  0.7bc 2.7  0.4a
Kokoti 252  6.1ab 9.6  0.4c 2.0  0.3ab
Lješkoplje 233  3.6c 10.1  0.7 ac 2.0  0.3ab
Nikolj Crkva 244  1.7bc 10.7  0.8ab 2.1  0.4ab
Kuci 255  2.6a 10.2  0.7 ac 2.0  0.4a
Mean 244  8.3A 10.3  0.8B 2.2  0.4C
Cabernet Sauvignon
Šipcanik 131  2.6a 12.5  0.5b 5.2  0.4a
Rogami 128  2.6a 13.1  0.5b 4.6  0.4ab
Kokoti 83  3.6d 15.8  0.8a 4.7  0.4ab
Lješkoplje 107  1.7b 15.9  0.9a 5.1  0.3ab
Nikolj Crkva 117  1.0bc 13.6  0.5b 5.1  0.3ab
Kuci 110  1.4b 15.4  0.4a 4.5  0.4b
Mean 113  16.4C 14.4  1.7A 4.9  0.4A

†Data are average values for 100 berries  SD. For each cultivar, values
followed by different lowercase letters (within a column) are significantly dif-
ferent (P < 0.05) by location. Mean values represent data averaged by loca-
tion and means followed by different uppercase letters (within a column) are
significantly different (P < 0.05) by cultivar. ‡Proportion of berry mass.
Figure 3. Factor scores for the first two principal components for Vranac
(VR, ), Kratošija (KR, ) and Cabernet Sauvignon (CS, ) cultivars grown
in Šipcanik (1), Rogami (2), Kokoti (3), Lješkoplje (4), Nikolj Crkva (5) and
Kuci (6) localities in Podgorica district.

© 2019 Australian Society of Viticulture and Oenology Inc.

6 Flavan-3-ols of Montenegrin grape cultivars
samples were clustered together on the left side of the scat-
ter plot as a consequence of a high proportion of catechin
and epicatechin in the seeds and skins, whereas Vranac
samples were located on the right side of the plot because of
the high proportion of epicatechin-3-O-gallate in the seeds
and procyanidin B2 in the skins. Kratošija samples were
found in the middle of the scatter plot, between Cabernet
Sauvignon and Vranac samples, because of their intermedi-
ate concentration of catechin, epicatechin and epicatechin-
3-O-gallate in grape seeds and procyanidin B2 in grape
skins.
Vranac showed variability in the vineyard locations of
Kokoti, Nikolj Crkva and Kuci, which reflected a higher pro-
portion of skin procyanidin B1. Kratošija showed the
greatest differences in flavan-3-ol composition between
vineyard locations, because of the higher proportion of cate-
chin and epicatechin in skins of grapes harvested at
Šipcanik, Lješkoplje and Nikolj Crkva and the higher pro-
portion of procyanidin B1 in grapes from vineyards at
Rogami, Kokoti and Kuci. Cabernet Sauvignon samples
exhibited the greatest homogeneity in the composition of
the flavan-3-ols among the six locations.
Impact of vineyard location on seed and skin flavan-3-ols
The results showed that the flavan-3-ol concentration of
seeds and skins varied significantly (P < 0.05) between the
six vineyard locations (Table 1). In seeds of all three culti-
vars, the concentration of all catechin monomers (catechin,
epicatechin and epicatechin-3-O-gallate), was higher at
Nikolj Crkva, which was 50 masl, while the lowest values
were found at the location with the highest altitude, Kuci,
which was 450 masl.
In skins of Vranac grapes, the concentration of catechin
monomers (catechin and epicatechin) and procyanidin B2
was uniform between locations, while differences in the
procyanidin B1 concentration were exhibited, with the
highest values observed at locations Kokoti (23 masl) and
Rogami (51 masl). Kratošija grape skins had the highest con-
centration of catechin and epicatechin at locations Šipcanik,
Lješkoplje and Nikolj Crkva. The procyanidin B1 concentra-
tion of skins was highest at Kokoti and Nikolj Crkva, while
procyanidin B2 concentration in skins was uniform among
locations. Cabernet Sauvignon skins exhibited a uniform con-
centration of catechin, epicatechin and procyanidin B2
between vineyard locations. There were differences in the
concentration of procyanidin B1, with a higher concentration
detected at Šipcanik, Rogami and Kuci. The highest concen-
tration of flavan-3-ols was found in berries of all cultivars
from Nikolj Crkva. Vranac had a high concentration of
flavan-3-ol at Lješkoplje, while the flavan-3-ol concentration
of Cabernet Sauvignon grapes was high at Kokoti, as well.
The location with the highest altitude, Kuci, gave the lowest
concentration of flavan-3-ol in berries for the three cultivars.
Discussion
In this study, we investigated the concentration of the
flavan-3-ols of skins and seeds of three red grape cultivars,
Vranac, Kratošija and Cabernet Sauvignon, sourced from six
vineyard locations in Montenegro.
Flavan-3-ols in seeds
The majority of flavan-3-ols found in grapes were detected
in the seeds for each cultivar (Table 1), which agreed with
previous research (Bourzeix et al. 1986, De Freitas et al.
2000, Souquet et al. 2000, Mattivi et al. 2009). The flavan-
Australian Journal of Grape and Wine Research 2019
3-ol concentration observed in seeds in the current study
(510–945 mg/kg) was in line with that reported in red
V. vinifera grape cultivars grown in the warm climate of
Castilla-La Mancha. Rodriguez Montealegre et al. (2006)
showed that the concentration of flavan-3-ol of seeds varied
between 300 and 870 mg/kg, with the highest concentration
reported for Cabernet Sauvignon and Merlot grapes. In con-
trast, the concentration of flavan-3-ol observed in this study
in cultivars grown in Montenegro was much lower than
that reported by Souquet et al. (2000) for red grape cultivars
grown in Bordeaux (1197–2323 mg/kg), an area with less
hot summers. Certainly, climatic factors can influence the
flavan-3-ol composition of seeds. Ivanova et al. (2011)
reported that the seeds of Vranac grapes grown in Macedo-
nia, contained a significantly lower amount of flavan-3-ol
than the seeds of Merlot grapes.
The berry mass for Vranac and Kratošija was almost twice
that of Cabernet Sauvignon, and these cultivars had a signifi-
cantly lower proportion of seed mass which corresponded to
a lower concentration of flavan-3-ol in the berries, because of
dilution. The physico-chemical characteristics of grape berries
had a notable influence on the concentration of flavan-3-ol
(Table 1), which was in line with results reported by
Rodriguez Montealegre et al. (2006), who found that berry
size indirectly affected the final concentration of phenolic
substances, whereas the number of seeds had a direct influ-
ence. Harbertson et al. (2002), however, studied Cabernet
Sauvignon, Syrah and Pinot Noir and found that the main
factor that contributed to the difference in seed tannin con-
centration was the number of seeds per berry.
The flavan-3-ols, catechin, epicatechin and epicatechin-
3-O-gallate, detected in the seeds of the cultivars studied,
were consistent with those reported in the literature
(De Freitas et al. 2000, Souquet et al. 2000, Mattivi et al.
2009, Návojská et al. 2012). In accordance with other studies
(Mateus et al. 2001, Monagas et al. 2003, Rodriguez
Montealegre et al. 2006, Chira et al. 2009), catechin was the
main flavan-3-ol present in seeds. After thioacidolysis, how-
ever, epicatechin is usually the main flavan-3-ol (Souquet
et al. 2000, Mattivi et al. 2009). The flavan-3-ol profile was
influenced by cultivar. The proportion of monomeric flavan-
3-ols (catechin, epicatechin and epicatechin-3-O-gallate) pre-
sent in Vranac seeds (i.e. 41/26/33%, respectively) cor-
responded to data published by Bogicevic (2009)
(i.e. 42/26/32%), who studied the same cultivar, also grown
in Montenegro. A considerable difference in the relative con-
centration of epicatechin-3-O-gallate was observed, with
Vranac and Kratošija having a higher concentration than
Cabernet Sauvignon. The relative concentration of
epicatechin-3-O-gallate found in Cabernet Sauvignon grown
in Montenegro (15.2%) was higher than that reported else-
where, being 5.8% (Rodriguez Montealegre et al. 2006),
9.4% (De Freitas et al. 2000), and 3.3 and 8.8% in two vin-
tages (Chira et al. 2009). This observation provides evidence
supporting the influence of environmental factors on the
flavan-3-ol profile of grapes, and possibly allows for classifica-
tion of cultivars by region of cultivation (i.e. the potential for
flavan-3-ols to be used as regional markers). The data pres-
ented here, together with data from the literature (Fuleki and
Ricardo da Silva 1997), suggest that the flavan-3-ol composi-
tion of grape seeds is greatly affected by cultivar.
Flavan-3-ols in skins
The flavan-3-ol concentration of skins observed in this study
(6.4–10.4 mg/kg) was lower than that reported by other
© 2019 Australian Society of Viticulture and Oenology Inc.
Pajovic-Šcepanovic et al.
researchers. Katalanic (1999) found that the total amount of
four skin flavan-3-ols was between 20 and 31 mg/kg. Sun
et al. (1999) measured a flavan-3-ol concentration of
33.8 mg/kg in Cabernet Sauvignon, whereas Rodriguez
Montealegre et al. (2006) reported a concentration between
62.9 and 91.4 mg/kg. The relationships observed among cul-
tivars and samples are similar to that observed in the current
study.
The finding that catechin was the main monomeric
flavan-3-ol present in skins was in accordance with previous
research (Bourzeix et al. 1986, Katalanic 1999, Sun et al.
1999, De Freitas et al. 2000). Additionally, procyanidin B1
was found to be the main dimer present in skins—with dif-
ferences between cultivars found—which is in agreement
with earlier research (Bourzeix et al. 1986, Katalanic 1999,
Sun et al. 1999, Mateus et al. 2001, Rodriguez Montealegre
et al. 2006). Cabernet Sauvignon berries had a higher pro-
portion of skin compared to that of Vranac and Kratošija
berries, an aspect reflected in the concentration of flavan-
3-ols in the berries (Table 1). The physico-chemical struc-
ture of berries, especially the proportion of skin, was found
to influence the flavan-3-ol concentration, which agrees
with the findings of Mattivi et al. (2006), who suggested
that thick-skinned berries such as Cabernet Sauvignon had
a higher concentration of flavan-3-ols than thin-skinned
berries of other cultivars.
The composition and concentration of flavan-3-ols in
the skins and seeds of the three cultivars studied was differ-
ent. Cabernet Sauvignon berries had a much higher flavan-
3-ol concentration compared to that of the Vranac and
Kratošija berries. This was in line with previous findings for
these three cultivars for the Montenegrin region. In a 2 year
study, Pajovic et al. (2014a) used a selective extraction
method which mimicked the vinification process and after-
wards analysed the concentration of low and high molecular
mass proanthocyanidins in seeds and skins using spectro-
photometry. The flavan-3-ol concentration reported by
Pajovic et al. (2014a) was much higher than in the current
study, which can be attributed to the methods of extraction
and analysis employed (Waterhouse et al. 2000). The find-
ing that Cabernet Sauvignon had the highest flavan-3-ol
concentration of the cultivars studied is in accordance with
previous studies from the wine regions of Bordeaux and
Navarra (De Freitas et al. 2000, Monagas et al. 2003). None-
theless, different climatic conditions apply in each region.
These results suggest that the concentration and composi-
tion of the flavan-3-ols of grape berries are dependent on
the cultivar, as reported in other previous studies (Monagas
et al. 2003, Chira et al. 2009, Mattivi et al. 2009).
Impact of vineyard location on seed and skin flavan-3-ols
Differences in grape composition, with respect to the flavan-
3-ol concentration of seeds and skins of the three cultivars,
were also found among different vineyard locations in the
Podgorica winegrowing area. The vineyards at Šipcanik,
Rogami, Kokoti, Lješkoplje and Nikolj Crkva lie between
27 and 51 masl, whereas the vineyard at Kuci is 450 masl.
The altitude of vineyards has been found to impact the con-
centration of flavan-3-ol of grape skins and seeds, with
lower altitude vineyard sites favouring the biosynthesis of
flavan-3-ol (Mateus et al. 2001). This finding was corrobo-
rated in the current study, with seeds of Vranac, Kratošija
and Cabernet Sauvignon grapes having the highest concen-
tration of catechin monomer when sourced from vineyards
with the lowest altitude (51 and 50 masl), and the lowest
© 2019 Australian Society of Viticulture and Oenology Inc.
Flavan-3-ols of Montenegrin grape cultivars 7
concentration at the highest altitude (454 masl). Most of the
flavan-3-ols measured in the skin (catechin, epicatechin,
procyanidin B1 and procyanidin B2) were present at higher
concentration at lower altitudes (27–51 masl), and at their
lowest concentration at the higher altitude (454 masl).
The grapes from the three cultivars grown in the Nikolj
Crkva vineyard location had the highest concentration of
flavan-3-ol, which agrees with a previous study involving
the monitoring of total extractable tannins in grapes from
several locations in the Podgorica vineyard district over
2 years (Pajovic et al. 2014a).
Conclusions
The present study contributed to the ongoing characterisa-
tion of the monomeric flavan-3-ols present in the seeds and
skins of autochthonous Montenegrin grape cultivars, Vranac
and Kratošija, as well as the internationally recognised culti-
var, Cabernet Sauvignon. Analysis of the monomeric
flavan-3-ols in seeds identified catechin as the most abun-
dant flavan-3-ol in each of the cultivars studied; with culti-
vars characterised by differences in their flavan-3-ol
profiles, that is the relative concentration of catechin, epi-
catechin and epicatechin-3-O-gallate. Vranac grapes were
found to contain a relatively high concentration of
epicatechin-3-O-gallate in their seeds. Grape skins contained
a low concentration of flavan-3-ol compared to seeds. Cate-
chin was the main monomeric flavan-3-ol present in the
skins of all cultivars, whereas procyanidin B1 was present at
a concentration higher than that of the other flavan-3-ols
analysed. The flavan-3-ol concentration of berries was
influenced by cultivar, with Cabernet Sauvignon > Kratošija
> Vranac. The berries of indigenous Montenegrin cultivars
had a significantly lower proportion of seed and skin mass
than Cabernet Sauvignon berries, which resulted in a lower
concentration of flavan-3-ol in the berries. The varietal
effect was also verified by multivariate analysis, which sepa-
rated cultivars according to their flavan-3-ol composition.
Geographic location was vital to the flavan-3-ol concentra-
tion of the three cultivars studied. It was concluded that at
lower altitude, the biosynthesis of flavan-3-ols is enhanced,
an aspect deserving further attention. Differentiation was
clearly demonstrated between cultivars on the basis of
flavan-3-ol composition of grape skins and seeds, thus it
should be possible to: (i) improve the current understanding
of the winemaking sector in Montenegro; and (ii) develop
tools and knowledge to enhance wine authenticity.
According to the type of wine desired, vineyard locations
can be chosen to enable differentiation of grapes, and there-
fore wines, based on flavan-3-ol composition.
Acknowledgements
This research was completed with support from a training
exchange grant from the project FP7-REGPOT-2010-5 and a
‘One Month Visits/Benedek’ scholarship donated by the
Austrian Agency for International Mobility and Cooperation
in Education, Science and Research OeAD, Austria. The
authors also thank the producers who provided the samples
studied, together with colleagues at the University of Mon-
tenegro, the Federal College and Research Institute for Viti-
culture and Pomology and University of Life Sciences, for
their help in the realisation of this study.
References
Bogicevic, M. (2009) Caratterizzazione varietale della cultivar
‘Vranac’, uva a bacca nera autoctona del Montenegro. Tesi della
8 Flavan-3-ols of Montenegrin grape cultivars
laureta magistrale. Master Thesis, Universita degli studi di Milano,
Milan, Italy.
Bourzeix, M., Weyland, D. and Heredia, N. (1986) Étude des cat-
échines et des procyanidols de la grape de raisin du vin et d’autres
dérivés de la vigne. Bulletin of the OIV 59, 1171–1252.
Božinovic, Z. (2005) Ampelografija [Ampelography] (Akademik:
Skopje, North Macedonia).
Chira, K., Schmauch, G., Saucier, C., Fabre, S. and Teissedre, P.L.
(2009) Grape variety effect on proanthocyanidin composition and
sensory perception of skin and seed tannin extracts from Bor-
deaux wine grapes (Cabernet Sauvignon and Merlot) for two con-
secutive vintages (2006 and 2007). Journal of Agricultural and
Food Chemistry 57, 545–553.
De Freitas, V.A.P., Glories, Y. and Monique, A. (2000) Developmen-
tal changes of procyanidins in grapes of red Vitis vinifera varieties
and their composition in respective wines. American Journal of
Enology and Viticulture 51, 397–403.
Downey, M.O., Dokoozlian, N.K. and Krstic, M.P. (2006) Cultural
practice and environmental impacts on the flavonoid composition
of grapes and wine: a review of recent research. American Journal
of Enology and Viticulture 57, 257–268.
Fuleki, T. and Ricardo da Silva, J.M. (1997) Catechin and procyanidin
composition of seeds from grape cultivars grown in Ontario. Journal
of Agricultural and Food Chemistry 45, 1156–1160.
Gris, E.F., Mattivi, F., Ferreira, E.A., Vrhovsek, U., Pedrosa, R.C.
and Bordignon-Luiz, M.T. (2011) Procyanidin profile and antioxi-
dant capacity of Brazilian Vitis vinifera red wines. Food Chemistry
44, 2851–2860.
Harbertson, J.F., Kennedy, J.A. and Adams, D.O. (2002) Tannin in
skins and seeds of Cabernet Sauvignon, Syrah, and Pinot Noir
berries during ripening. American Journal of Enology and Viticul-
ture 53, 54–59.
Ivanova, V., Stefova, M., Vojnoski, B., Dörnyei, A.,  Márk, L.,
Dimovska, V., Stafilov, T. and Kilár, F. (2011) Identification of
polyphenolic compounds in red and white grape varieties grown
in R. Macedonia and changes of their content during ripening.
Food Research International 44, 2851–2860.
Katalanic, V. (1999) Grape catechins—natural antioxidants. Journal
of Wine Research 10, 15–23.
Liszt, K., Eder, R., Wendelin, S. and Somoza, V. (2015) Identifica-
tion of catechin, syringic acid, and procyanidin B2 in wine as stim-
ulants of gastric acid secretion. Journal of Agricultural and Food
Chemistry 63, 7775–7783.
Mateus, N., Marques, S., Goncalves, A.C., Machado, J.M. and De
Freitas, V. (2001) Proanthocyanidin composition of red Vitis vinifera
varieties from the Douro Valley during ripening: influence of cultiva-
tion altitude. American Journal of Enology and Viticulture 52,
115–121.
Mattivi, F., Vrhovšek, U., Masuero, D. and Trainotti, D. (2009) Dif-
ferences in the amount and structure of extractable skin and seed
tannins amongst red grape varieties. Australian Journal of Grape
and Wine Research 15, 27–35.
Mattivi, F., Guzzon, R., Vrhovšek, U., Stefanini, M. and Velasco, R.
(2006) Metabolite profiling of grape: flavonols and anthocyanins.
Journal of Agricultural and Food Chemistry 54, 7692–7702.
Monagas, M., Gómez-Cordovés, C., Bartolomé, B., Laureano, O.
and Ricardo da Silva, J.M. (2003) Monomeric, oligomeric, and
polymeric flavan-3-ol composition of wines and grapes from Vitis
vinifera L. cv. Graciano, Tempranillo, and Cabernet Sauvignon.
Journal of Agricultural and Food Chemistry 51, 6475–6481.
Návojská, J., Wendelin, S., Eder, R. and Francáková, H. (2012)
Identification of oenological tannins extracted from oak wood.
Mitteilungen Klosterneuburg 64, 9–19.
Organisation Internationale de la Vigne et du Vin (2009) Oenologi-
cal tannins. International oenological codex (Organisation Inter-
nationale de la Vigne et du Vin: Paris, France) pp. 5–13.
Australian Journal of Grape and Wine Research 2019
Pajovic, R., Wendelin, S., Forneck, A. and Eder, R. (2014a) Varietal
differentiation of grapes cv. ‘Vranac’, ‘Kratošija’ and ‘Cabernet
Sauvignon’ from Montenegro according to their polyphenolic
composition. Mitteilungen Klosterneuburg 64, 9–19.
Pajovic, R., Raicevic, D., Popovic, T., Sivilotti, P., Lisjak, K. and
Vanzo, A. (2014b) Polyphenolic characterisation of Vranac,
Kratošija and Cabernet Sauvignon (Vitis vinifera L. cv.) grapes and
wines from different vineyard locations in Montenegro.
South African Journal of Enology and Viticulture 35, 139–148.
Pajovic-Šcepanovic, R., Wendelin, S. and Eder, R. (2018) Phenolic
composition and varietal discrimination of Montenegrin red wines
(Vitis vinifera var. Vranac, Kratošija, and Cabernet Sauvignon).
European Food Research and Technology 244, 2243–2254.
Peleg, H., Gacon, K., Schlich, P. and Noble, A.C. (1999)
Bitterness and astringency of flavan-3-ol monomers, dimers and
trimers. Journal of the Science of Food and Agriculture 79,
1123–1128.
Rigo, A., Vianello, F., Clementi, G., Rossetto, M., Scarpa, M. and
Vrhovšek, U. (2000) Contribution of the procyanidins to the per-
oxy radical scavenging capacity of some Italian red wines. Journal
of the Science of Food and Agriculture 48, 1996–2002.
Rodriguez Montealegre, R., Romero Peces, R., Chacon
Vozmediano, J.L., Martiez Gascuena, J. and Garcia Romero, E.
(2006) Phenolic compounds in skins and seeds of ten grape Vitis
vinifera varieties grown in a warm climate. Journal of Food Com-
position and Analysis 19, 687–693.
Somers, T.C. (1971) The polymeric nature of wine pigments. Phyto-
chemistry 10, 2175–2186.
Souquet, J.M., Cheynier, V. and Moutounet, M. (2000) Les pro-
antocyanidines du raisin. Bulletin of the OIV 835–836, 601–609.
Souquet, J.M., Cheynier, V., Brossaud, F. and Moutounet, M.
(1996) Polymeric procyanidins from grape skins. Phytochemistry
43, 509–512.
Sun, B.S., Pinto, T., Leandro, M.C., Ricardo-da-Silva, J.M. and
Spranger, M.I. (1999) Transfer of catechins and procyanidins from
solid parts of the grape cluster into wine. American Journal of
Enology and Viticulture 50, 179–184.
Tonietto, J. and Carbonneau, A. (2004) A multicriteria climatic clas-
sification system for grape-growing regions worldwide. Agricul-
tural and Forest Meteorology 124, 81–97.
Ulicevic, M. (1966) Prilog proucavanju osobina najvažnijih sorata
vinove loze gajenih u SR Crnoj Gori. Arhiv za poljoprivredne
nauke [Contribution to characterization of the most important
grapevine varieties grown in Republic of Montenegro]. Archives
of Agricultural Sciences 23, 1–100.
Vrhovšek, U., Wendelin, S. and Eder, R. (1997) Quantitative
Bestimmung von Hydroxyzimtsäuren und hydroxyzimtsäurederivaten
(Hydroxycinnamaten) in Weißweinen mittels HPLC.
Mitteilungen Klosterneuburg 47, 164–172.
Vujadinovic, M., Vukovic, A., Jakšic, D., Pavicevic, S., Mijanovic, T.,
Pažin, N., Drljevic, M., Popovic, T., Beader, M., Perovic, V.,
Ivaniševic, D., Maraš, V. and La Notte, P. (2016) Bioclimatic viti-
cultural indices in Montenegro. Gregori, J. and Doran, N., eds.
Proceedings of the 11th international terroir congress 2016; 10–14
July 2016; McMinnville, OR, USA (Southern Oregon University:
Ashland, OR, USA) pp. 117–117.
Waterhouse, A.L., Ignelzi, S. and Shirley, J.S. (2000) A comparison
of methods for quantifying oligomeric procyanidins from grape
seed extracts. American Journal of Enology and Viticulture 51,
383–389.
Manuscript received: 24 July 2018
Revised manuscript received: 11 February 2019
Accepted: 9 April 2019
© 2019 Australian Society of Viticulture and Oenology Inc.